Substrate proteolysis is inhibited by dominant-negative Nedd4 and Rsp5 mutants harboring alterations in WW domain 1

Mammalian Nedd4 and its budding yeast orthologue Rsp5 are members of a large family of HECT-domain-containing ubiquitin ligases. Besides possessing a Ca2+/lipid-binding domain, both ligases have multiple protein-interacting modules termed WW domains. The C-terminal WW domains mediate interactions with substrates, but the function of the first WW domain remains unclear. We found that expression of a WW domain 1 Nedd4 mutant inhibits the growth of budding yeast by affecting the rsp5-ole1 pathway. The WW domain 1 mutant-induced phenotype is suppressed by ole1 cDNA overexpression or oleic acid supplementation of growth media and ole1 RNA levels are reduced in cells expressing this Nedd4 mutant. Also, the WW domain 1 Nedd4 mutant associates via WW domains 2 and 3 with Spt23, a Rsp5 target and ole1 transactivator. The dominant-negative activity of this mutant is associated with promoting accumulation of unprocessed Spt23 and inhibiting generation of processed and presumably active protein. Also, Spt23 processing is inhibited by a Nedd4 mutant that lacks ubiquitin ligase activity and Spt23-binding-competent Rsp5 mutants harboring WW domain 1 or ligase domain mutations. Interestingly, in mammalian cells, wild-type Nedd4 promotes proteasome-mediated degradation of the precursor form of Spt23. WW domain 1 and ligase domain Nedd4 mutants block its degradation. These results indicate that WW domain 1 of these ligases interacts with cofactors that are required for ubiquitin/proteasome-dependent proteolysis of bound substrates.Natalia Shcherbik, Sharad Kumar and Dale S. Haine

A co-cretion process for premium traditional portuguese pocket knifes

During an eight weeks DEMOLA project, from March to June 2019, a team of five students together with the representative of Martins Cutelaria Tradicional de Palaçoulo, putted into practice the steps towards an innovative design of a new premium series, the “Portuguese History Collection”. This design project was driven through a co-creation process with the traditional Portuguese pocket knifes company Martins Cutelaria Tradicional de Palaçoulo, based in the old village of Palaçoulo, Bragança, Portugal (see [1]). Martins Cutelaria, which has a long history in the design, production and innovation of traditional Portuguese pocket knifes, launched the challenge of designing a new premium line for their traditional pocket knifes segment. The challenge was to innovate while keeping untouched the identity, tradition and the solid values that are the pillars of the company since it was founded in 1954. Based on the company's motto: “Ter uma navalha Martins Palaçoulo, é ter nas mãos um pedaço da nossa história."(*) and inspired by the fact that the year 2019 celebrates the 500th anniversary of the departure of the navigator Fernão de Magalhães for his circumnavigation of the globe by sea, the team worked out the new pocket knifes collection “Portuguese History Collection – The Navigations”.info:eu-repo/semantics/publishedVersio

Endocytic regulation of alkali metal transport proteins in mammals, yeast and plants

The relative concentrations of ions and solutes inside cells are actively maintained by several classes of transport proteins, in many cases against their concentration gradient. These transport processes, which consume a large portion of cellular energy, must be constantly regulated. Many structurally distinct families of channels, carriers, and pumps have been characterized in considerable detail during the past decades and defects in the function of some of these proteins have been linked to a growing list of human diseases. The dynamic regulation of the transport proteins present at the cell surface is vital for both normal cellular function and for the successful adaptation to changing environments. The composition of proteins present at the cell surface is controlled on both the transcriptional and post-translational level. Post-translational regulation involves highly conserved mechanisms of phosphorylation- and ubiquitylation-dependent signal transduction routes used to modify the cohort of receptors and transport proteins present under any given circumstances. In this review, we will summarize what is currently known about one facet of this regulatory process: the endocytic regulation of alkali metal transport proteins. The physiological relevance, major contributors, parallels and missing pieces of the puzzle in mammals, yeast and plants will be discussed.This work was supported by grant BFU2011-30197-C03-03 from the Ministerio de Ciencia e Innovacion (Spain). V.L.-T. is supported by a fellowship from the Universidad Politecnica de Valencia. C. P. is supported by a fellowship from the Consejo Superior de Investigaciones Cientificas (Spain).Mulet Salort, JM.; Llopis Torregrosa, V.; Primo Planta, C.; Marques Romero, MC.; Yenush, L. (2013). 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Implementation of new approaches for generating conventional reassortants for live attenuated influenza vaccine based on Russian master donor viruses

Cold-adapted influenza strains A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69, originally developed in Russia, have been reliable master donors of attenuation for preparing live attenuated influenza vaccines (LAIV). The classical strategy for generating LAIV reassortants is robust, but has some disadvantages. The generation of reassortants requires at least 3 passages under selective conditions after co-infection; each of these selective passages takes six days. Screening the reassortants for a genomic composition traditionally starts after a second limiting dilution cloning procedure, and the number of suitable reassortants is limited. We developed a new approach to shorten process of preparing LAIV seed viruses. Introducing the genotyping of reassortants by pyrosequencing and monitoring sequence integrity of surface antigens starting at the first selective passage allowed specific selection of suitable reassortants for the next cloning procedure and also eliminate one of the group selective passage in vaccine candidate generation. Homogeneity analysis confirmed that reducing the number of selective passages didn't affect the quality of LAIV seed viruses. Finally, the two-way hemagglutination inhibition test, implemented for all the final seed viruses, confirmed that any amino acid substitutions acquired by reassortants during egg propagation didn't affect antigenicity of the vaccine. Our new strategy reduces the time required to generate a vaccine and was used to generate seasonal LAIVs candidates for the 2012/2013, 2014/2015, and 2015/2016 seasons more rapidly. © 2015

Application of real time RT-PCR for the genetic homogeneity and stability tests of the seed candidates for live attenuated influenza vaccine production

Development and improvement of quality control tests for live attenuated vaccines are a high priority because of safety concerns. Live attenuated influenza vaccine (LAIV) viruses are 6:2 reassortants containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from circulating influenza viruses to induce protective immune responses, and the six internal gene segments from a cold-adapted Master Donor Virus (MDV). LAIV candidate viruses for the 2012-2013 seasons, A/Victoria/361/2011-CDC-LV1 (LV1) and B/Texas/06/2011-CDC-LV2B (LV2B), were created by classical reassortment of A/Victoria/361/2011 and MDV-A A/Leningrad/134/17/57 (H2N2) or B/Texas/06/2011 and MDV-B B/USSR/60/69. In an attempt to provide better identity and stability testing for quality control of LV1 and LV2B, sensitive real-time RT-PCR assays (rRT-PCR) were developed to detect the presence of undesired gene segments (HA and NA from MDV and the six internal genes from the seasonal influenza viruses). The sensitivity of rRT-PCR assays designed for each gene segment ranged from 0.08 to 0.8EID50 (50% of Egg Infectious Dose) per reaction for the detection of undesired genes in LV1 and from 0.1 to 1EID50 per reaction for the detection of undesired genes in LV2B. No undesired genes were detected either before or after five passages of LV1 or LV2B in eggs. The complete genome sequencing of LV1 and LV2B confirmed the results of rRT-PCR, demonstrating the utility of the new rRT-PCR assays to provide the evidence for the homogeneity of the prepared vaccine candidate. © 2013

Evaluation of A(H1N1)pdm09 LAIV vaccine candidates stability and replication efficiency in primary human nasal epithelial cells

The recent reduction of live attenuated influenza vaccine (LAIV) effectiveness in multivalent formulations was particularly associated with the A(H1N1)pdm09 component. In the 2017 the WHO vaccine composition committee changed its recommendations for the A(H1N1)pdm09 component to include an A/Michigan/45/2015-like virus. We evaluated effectiveness and quality of newly developed and previous A(H1N1)pdm09 LAIV reassortants through assessment of their thermal and pH stability, receptor binding specificity and replication fitness in primary human airway epithelial cells of nasal origin (hAECN). Our analysis showed that LAIV expressed hemagglutinin (HA) and neuraminidase (NA) from an A/Michigan/45/2015-like strain A/New York/61/2015 (A/New York/61/2015-CDC-LV16A, NY-LV16A), exhibit higher thermal and pH stability compared to the previous vaccine candidates expressing HA and NA from A/California/07/2009 and A/Bolivia/559/2013 (A17/Cal09 and A17/Bol13). Reassortants A/South Africa/3626/2013-CDC-LV14A (SA-LV14A) and NY-LV16A showed preferential binding to α2,6 sialic acid (SA) receptors and replicated at higher titers and more extensively in hAECN compared to A17/Cal09 and A17/Bol13, which had an α2,3 SA receptor binding preference. Our data analysis supports selection of A/New York/61/2015-CDC-LV16A for LAIV formulation and the introduction of new assays for LAIV characterization. © 2019 The Author(s

Rsp5p Is Required for ER Bound Mga2p120 Polyubiquitination and Release of the Processed/Tethered Transactivator Mga2p90

AbstractA number of eukaryotic transcription factors are held in a latent state by being embedded in, or tethered to, cellular membranes. Mga2p of Saccharomyces cerevisiae is an endoplasmic reticulum (ER)-localized transcription factor that plays an overlapping role with homologous Spt23p in upregulating expression of OLE1, a gene required for the synthesis of essential oleic acid [1]. Previous studies have documented that proteasome-dependent processing of ER bound 120 kDa Mga2p and Spt23p proteins generates transcriptionally competent 90 kDa polypeptides [2]. In the case of Spt23p90, it is held at the membrane prior to release via a self-interaction with the unprocessed Spt23p120 anchor [3]. It is currently thought that the highly conserved Rsp5p ubiquitin ligase provides the signal for partial degradation of both proteins. Cells lacking Rsp5p function require oleic acid for growth, and Spt23p processing is suppressed in rsp5Δ cells and in wild-type RSP5 cells upon expression of Rsp5p dominant-negative mutants [2, 4]. We report here that Rsp5p is dispensable for Mga2p90 generation but not for release of the processed product from the ER. In addition, we demonstrate that polyubiquitinated Mga2p120 accumulates in cells lacking Npl4p or proteasome function and Rsp5p is required for Mga2p120 polyubiquitination. Finally, we provide evidence that Mga2p90 and Mga2p120 dimerize and that Rsp5p binds heterodimeric Mga2p complexes both in vitro and in vivo. In light of these experiments, we propose that Rsp5p facilitates Mga2p90 release from the ER by promoting polyubiquitination and Npl4p-proteasome-mediated degradation of the interacting Mga2p120 ER bound anchor